Interpretive Summary: Some bacteria in the genus Rhodococcus cause leafy galls, shoot proliferation, and other growth deformities on many plants and are economically important pathogens of herbaceous perennials grown by the ornamental nursery industry. Little is known about this bacterial pathogen, and this dearth of knowledge impedes efforts by nursery growers to detect and manage the pathogen. This manuscript describes the genomic sequences of 20 isolates of Rhodococcus isolated from herbaceous perennials exhibiting symptoms of leafy gall. Comparisons among the genomes revealed a high level of diversity among the isolates that cause disease. These comparisons also showed that a specific cytokinin plant hormone is associated with virulence of these bacteria. These data provide the first genomic comparison in this group of plant pathogenic bacteria and lay the groundwork needed to develop diagnostic tools needed by the nursery industry.

Technical Abstract:
Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one cytokinin type that was specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse clade of Rhodococcus.